Root system analysis is a complex task, often performed with fully automated image analysis pipelines. However, the outcome is rarely verified by ground-truth data, which might lead to underestimated ... [more ▼]

Root system analysis is a complex task, often performed with fully automated image analysis pipelines. However, the outcome is rarely verified by ground-truth data, which might lead to underestimated biases. We have used a root model, ArchiSimple, to create a large and diverse library of ground-truth root system images (10,000). For each image, three levels of noise were created. This library was used to evaluate the accuracy and usefulness of several image descriptors classically used in root image analysis softwares. Our analysis highlighted that the accuracy of the different traits is strongly dependent on the quality of the images and the type, size and complexity of the root systems analysed. Our study also demonstrated that machine learning algorithms can be trained on a synthetic library to improve the estimation of several root system traits. Overall, our analysis is a call to caution when using automatic root image analysis tools. If a thorough calibration is not performed on the dataset of interest, unexpected errors might arise, especially for large and complex root images. To facilitate such calibration, both the image library and the different codes used in the study have been made available to the community. [less ▲]

In the era that huge numbers of microbial genomes are being released in the databases, it becomes increasingly important to rapidly mine genes as well as predict the regulatory networks that control their ... [more ▼]

In the era that huge numbers of microbial genomes are being released in the databases, it becomes increasingly important to rapidly mine genes as well as predict the regulatory networks that control their expression. To this end, we have developed an improved and online version of the PREDetector software aimed at identifying putative transcription factor-binding sites (TFBS) in bacterial genomes. The original philosophy of PREDetector 1.0 is maintained, i.e. to allow users to freely fix the DNA-motif screening parameters, and to provide a statistical means to estimate the reliability of the prediction output. This new version offers an interactive table as well as graphics to dynamically alter the main screening parameters with automatic update of the list of identified putative TFBS. PREDetector 2.0 also has the following additional options: (i) access to genome sequences from different databases, (ii) access to weight matrices from public repositories, (iii) visualization of the predicted hits in their genomic context, (iv) grouping of hits identified in the same upstream region, (v) possibility to store the performed jobs, and (vi) automated export of the results in various formats. PREDetector 2.0 is available at http://predetector.fsc.ulg.ac.be/. [less ▲]

Root system analysis is a complex task, often performed using fully automated image analysis pipelines. However, these pipelines are usually evaluated with a limited number of ground-truth root images ... [more ▼]

Root system analysis is a complex task, often performed using fully automated image analysis pipelines. However, these pipelines are usually evaluated with a limited number of ground-truth root images, most likely of limited size and complexity. We have used a root model, ArchiSimple to create a large and diverse library of ground-truth root system images (10.000). This library was used to evaluate the accuracy and usefulness of several image descriptors classicaly used in root image analysis pipelines. Our analysis highlighted that the accuracy of the different metrics is strongly linked to the type of root system analyzed (e.g. dicot or monocot) as well as their size and complexity. Metrics that have been shown to be accurate for small dicot root systems might fail for large dicots root systems or small monocot root systems. Our study also demonstrated that the usefulness of the different metrics when trying to discriminate genotypes or experimental conditions may vary. Overall, our analysis is a call to caution when automatically analyzing root images. If a thorough calibration is not performed on the dataset of interest, unexpected errors might arise, especially for large and complex root images. To facilitate such calibration, both the image library and the different codes used in the study have been made available to the community. [less ▲]

Molecular data concerning the involvement of roots in the genetic pathways regulating floral transition are lacking. In this study, we performed global analyses of the root transcriptome in Arabidopsis in ... [more ▼]

Molecular data concerning the involvement of roots in the genetic pathways regulating floral transition are lacking. In this study, we performed global analyses of the root transcriptome in Arabidopsis in order to identify flowering time genes that are expressed in the roots and genes that are differentially expressed in the roots during the induction of flowering. Data mining of public microarray experiments uncovered that about 200 genes whose mutations are reported to alter flowering time are expressed in the roots (i.e. were detected in more than 50% of the microarrays). However, only a few flowering integrator genes passed the analysis cutoff. Comparison of root transcriptome in short days and during synchronized induction of flowering by a single 22-h long day revealed that 595 genes were differentially expressed. Enrichment analyses of differentially expressed genes in root tissues, gene ontology categories, and cis-regulatory elements converged towards sugar signaling. We concluded that roots are integrated in systemic signaling, whereby carbon supply coordinates growth at the whole plant level during the induction of flowering. This coordination could involve the root circadian clock and cytokinin biosynthesis as a feed forward loop towards the shoot. [less ▲]

Manipulating plant architecture is key to increase crop yield. In this perspective, basic knowledge on the molecular mechanisms regulating plant growth and development will be fundamental for the future ... [more ▼]

Manipulating plant architecture is key to increase crop yield. In this perspective, basic knowledge on the molecular mechanisms regulating plant growth and development will be fundamental for the future of agriculture. Recent progress indicates that a family of plant genes, homologous to POSPHADITYLETHANOLAMINE-BINDING PROTEINS (PEBP) in other eukaryotes, plays critical roles in controlling shoot development traits such as branching, flowering, bud dormancy or tuberization. Very little is known however on the functions of these genes in root development. The current research will attempt to answer this question in the case of Cichorium intybus, a biannual plant cultivated for the extraction of inulin (a polymer of fructose) from its root. Functional analyses will include expression kinetics, complementation tests in Arabidopsis mutants and creation of PEBP- overexpressing or silencing chicory plants. The preliminary steps of the project are the identification of PEBP genes in chicory by in silico analyses, the determination of the time of the storage root initiation and the establishment of a regeneration protocol for the genetic transformation. [less ▲]

Chlorophyll fluorescence is an information-rich signal which provides an access to the management of light absorbed by PSII. A good example of this is the succession of fast fluorescence fluctuations ... [more ▼]

Chlorophyll fluorescence is an information-rich signal which provides an access to the management of light absorbed by PSII. A good example of this is the succession of fast fluorescence fluctuations during light-induced photosynthetic induction after dark-adaptation. During this period, the fluorescence trace exhibits several inflexion points: O-J-I-P-S-M-T. Thereas the OJIP part of this kinetics has been the subject of many studies, the processes that underly the PSMT transient are less understood. Here, we report an analysis of the PSMT phase in the green microalga Haematococcus pluvialis in terms of electron acceptors and light use by photochemistry, fluorescence and non-photochemical quenching (NPQ). We identify additional sub-phases between P and S delimited by an inflexion point, that we name Q, found in the second time-scale. The P-Q phase expresses a transient photochemical quenching specifically due to alternative electron transport to oxygen. During the transition from Q to S, the NPQ increases and then relaxes during the S-M phase in about 1 min. It is suggested that this transient NPQ observed during induction is a high energy state quenching (qE) dependent the alternative electron transport to molecular oxygen. We further show that this NPQ is of the same nature than the NPQ, known as the low-wave phenomenon, which is transiently observed after a saturating light pulse given at steady-state. In both cases, the NPQ is oxygen-dependent. This NPQ is observed at external pH 6.0, but not at pH 7.5, which seems correlated with faster saturation of the PQ pool at pH 6.0. [less ▲]

Plant-based biopharmaceuticals have gained a lot of interest in the past decade due to their reduced cost and relative safety compared to mammalian cell cultures. While the first plant-made recombinant ... [more ▼]

Plant-based biopharmaceuticals have gained a lot of interest in the past decade due to their reduced cost and relative safety compared to mammalian cell cultures. While the first plant-made recombinant proteins are now reaching the market, the production systems still need improvements to maximize their competitiveness. Optimizing production hosts requires the identification and subsequent inhibition of the most active endogenous peptidases, proteolysis being one of the main factors limiting yields. The aim of our study was to identify root-secreted proteases of Arabidopsis thaliana involved in target protein degradation (BSA) and inhibit them in vivo. Biochemical analyses identified serine proteases as the main class responsible for BSA degradation. An RT-qPCR experiment led to the choice of the serine protease gene SBT4.12 and its homologs as targets for an amiRNA-mediated silencing approach. Arabidopsis amiRNA-expressing lines showed lower levels of expression for SBT4.12 and reduced proteolytic activity in their rhizosecreted extracts. Crossing these lines with recombinant protein producing lines could lead to an improved production platform for proteins of interest. [less ▲]

Flowering is a hot topic in Plant Biology and important progress has been made in Arabidopsis thaliana toward unravelling the genetic networks involved. The increasing complexity and the explosion of ... [more ▼]

Flowering is a hot topic in Plant Biology and important progress has been made in Arabidopsis thaliana toward unravelling the genetic networks involved. The increasing complexity and the explosion of literature however require development of new tools for information management and update. We therefore created an evolutive and interactive database of flowering time genes, named FLOR-ID (Flowering-Interactive Database), that is available freely at http://www.flor-id.org. The hand-curated database contains information on 306 genes and links to 1595 publications gathering the work of more than 4500 authors. Gene function and interactions within the flowering pathways were inferred from the analysis of related publications, included in the database and translated into interactive manually drawn snapshots. [less ▲]

Plant-based biopharmaceuticals have gained a lot of interest in the past decade due to their reduced cost and relative safety compared to mammalian cell cultures. While the first plant-made recombinant ... [more ▼]

Plant-based biopharmaceuticals have gained a lot of interest in the past decade due to their reduced cost and relative safety compared to mammalian cell cultures. While the first plant-made recombinant proteins are now reaching the market, the production systems still need improvements to maximize their competitiveness, proteolysis being one of the main factors limiting the yields. Identifying and inhibiting in vivo endogenous proteases involved in the degradation of recombinant proteins could then lead to a significant increase in production yields. In this study, we focused on two different production systems in Arabidopsis thaliana: rhizosecretion and cell suspensions. Extracellular proteases of both systems were used in vitro to study the conditions of target protein degradation (Bovine Serum Albumine, BSA). First, proteases from both systems degrade BSA at both acidic and neutral-to-basic pH conditions. Then, serine and metallopeptidases were shown to be the main protease classes responsible for BSA degradation by rhizosecreted proteomes or extracellular cell culture media, respectively. Finally, the biochemical tests were coupled to a bioinformatics analysis of publicly available transcriptomic data, in order to reduce the number of the proteases most likely involved in BSA degradation. Using this method, only five serine proteases and two metallopeptidases remain candidates for an amiRNA-mediated in vivo inhibition. [less ▲]

The transition to flowering is an essential step of the plant life cycle that is tightly controlled by both endogenous and environmental cues. Its regulation is extremely complex and involves hundreds of ... [more ▼]

The transition to flowering is an essential step of the plant life cycle that is tightly controlled by both endogenous and environmental cues. Its regulation is extremely complex and involves hundreds of genes that are part of highly interconnected pathways. Our knowledge of the molecular mechanisms governing the floral induction of Arabidopsis thaliana increases quickly and a significant number of reviews are published every year on this topic. However, most of them focus on a single pathway without highlighting the interconnections existing between them. Furthermore, those reviews become rapidly outdated, since our comprehension of the genetic control of flowering time evolves continuously. Hence, we believe that the current landscape of flowering time research in Arabidopsis misses an exhaustive repository of the genes involved in the control of flowering and their regulatory pathways. Here, we present a new interactive resource built around a curated database of the flowering time genes that brings together multiple pieces of information such as their function, the flowering time phenotype of mutants and overexpressing lines, the related key publications, etc. Our website thus gives access to a curated and exhaustive list of the genes involved in the regulation of flowering time in Arabidopsis as well as the regulatory pathways controlling their expression. Because of its flexibility, the database is highly dynamic and will be periodically updated with the future breakthroughs in this domain. [less ▲]

Well-developed and functional roots are critical to support plant life and reach high crop yields. Their study however, is hampered by their underground growth and characterizing complex root system ... [more ▼]

Well-developed and functional roots are critical to support plant life and reach high crop yields. Their study however, is hampered by their underground growth and characterizing complex root system architecture therefore remains a challenge. In the model plant Arabidopsis thaliana, in vitro culture remains the easiest and preferred method to study root development, which limits the analyses to young seedlings. We present here an innovative design of hydroponic rhizotron (rhizoponics) designed for the root system analysis of adult plants of Arabidopsis thaliana. [less ▲]

We present here an innovative design of hydroponic rhizotrons (rhizoponics) adapted to Arabidopsis thaliana. The setup allows to simultaneously characterize the RSA and shoot development from seedling to ... [more ▼]

We present here an innovative design of hydroponic rhizotrons (rhizoponics) adapted to Arabidopsis thaliana. The setup allows to simultaneously characterize the RSA and shoot development from seedling to adult stages, i.e. from seed to seed. This system offers the advantages of hydroponics such as control of root environment and easy access to the roots for measurements or sampling. Being completely movable and low cost, it can be used in controlled cabinets. We chose the case of cadmium treatment to illustrate potential applications, from cell to organ levels. [less ▲]

Plant-based recombinant protein production systems have gained an extensive interest over the past few years, because of their reduced cost and relative safety. Although the first products are now ... [more ▼]

Plant-based recombinant protein production systems have gained an extensive interest over the past few years, because of their reduced cost and relative safety. Although the first products are now reaching the market, progress are still needed to improve plant hosts and strategies for biopharming. Targeting recombinant proteins toward the extracellular space offers several advantages in terms of protein folding and purification, but degradation events are observed, due to endogenous peptidases. This paper focuses on the analysis of extracellular proteolytic activities in two production systems: cell cultures and root-secretion (rhizosecretion), in Arabidopsis thaliana and Nicotiana tabacum. Proteolytic activities of extracellular proteomes (secretomes) were evaluated in vitro against two substrate proteins: bovine serum albumin (BSA) and human serum immunoglobulins G (hIgGs). Both targets were found to be degraded by the secretomes, BSA being more prone to proteolysis than hIgGs. The analysis of the proteolysis pH-dependence showed that target degradation was mainly dependent upon the production system: rhizosecretomes contained more peptidase activity than extracellular medium of cell suspensions, whereas variations due to plant species were smaller. Using class-specific peptidase inhibitors, serine and metallopeptidases were found to be responsible for degradation of both substrates. An in-depth in silico analysis of genomic and transcriptomic data from Arabidopsis was then performed and led to the identification of a limited number of serine and metallo-peptidases that are consistently expressed in both production systems. These peptidases should be prime candidates for further improvement of plant hosts by targeted silencing. [less ▲]

The in silico prediction of cis-acting elements in a genome is an efficient way to quickly obtain an overview of the biological processes controlled by a trans-acting factor, and connections between ... [more ▼]

The in silico prediction of cis-acting elements in a genome is an efficient way to quickly obtain an overview of the biological processes controlled by a trans-acting factor, and connections between regulatory networks. Several regulon prediction web tools are available, designed to identify DNA motifs predicted to be bound by transcription factors using position weight matrix-based algorithms. In this paper we expose and discuss the conflicting objectives of software creators (bioinformaticians) and software users (biologists), who aim for reliable and exhaustive prediction outputs, respectively. Software makers, concerned with providing tools that minimise the number of false positive hits, often impose a stringent threshold score for a sequence to be included in the list of the putative cis-acting sites. This rigidity eventually results in the identification of strongly reliable but largely straightforward sites, i.e. those associated with genes already anticipated to be targeted by the studied transcription factor. Importantly, this biased identification of strongly bound sequences contrasts with the biological reality where, in many circumstances, a weak DNA-protein interaction is required for the appropriate gene's expression. We show here a series of transcriptionally controlled systems involving weakly bound cis-acting elements that could never have been discovered because of the policy of preventing software users from modifying the screening parameters. Proposing only trustworthy prediction outputs thus prevents biologists from fully utilising their knowledge background and deciding to analyse statistically irrelevant hits that could nonetheless be potentially involved in subtle, unexpected, though essential cis-trans relationships. [less ▲]

Flowering is a crucial step in plant life cycle and is therefore tightly controlled by both environmental and endogenous cues. The involvement of the aerial organs of the plant in the molecular mechanisms ... [more ▼]

Flowering is a crucial step in plant life cycle and is therefore tightly controlled by both environmental and endogenous cues. The involvement of the aerial organs of the plant in the molecular mechanisms controlling floral transition has been extensively documented while the participation of the roots remains poorly investigated. However, the induction of flowering by photoperiod involves systemic signals that move in the phloem from leaves to sinks, and hence presumably reach the roots. We therefore performed a transcriptomic analysis of the roots during the induction of flowering in Arabidopsis thaliana and indeed identified a large number of differentially expressed genes. A reverse genetic approach further confirmed the pleiotropic effects of flowering time genes on root architecture. [less ▲]

Proteases are involved in many physiological processes during the whole life of the plant, such as embryonic development, defense against pathogens, nutrition or mycorrhiza creation. However, the ... [more ▼]

Proteases are involved in many physiological processes during the whole life of the plant, such as embryonic development, defense against pathogens, nutrition or mycorrhiza creation. However, the functions of many of the 800 proteases of Arabidopsis thaliana still remain unknown. Besides discovering new functions, studying proteases can also result in improving plant biotechnology. Indeed, plants can be used as hosts for recombinant protein production. Some proteins of interest require to be secreted in order to fold properly, but production yields are limited due to their degradation by endogenous extracellular proteases. The aim of our study is to identify active root-secreted proteases of Arabidopsis thaliana. Their activity was first analyzed by in vitro incubation with a target protein (BSA) at different values of pH and in the presence of proteases inhibitors. This analysis identified serine proteases as the major protease class involved in BSA degradation. Then, an Activity-Based Protein Profiling approach led to the labeling of two active serine proteases in the root-secreted sample. Finally, a further step towards the identification by mass spectrometry, based on affinity purification, was developed. [less ▲]